The goal of this R01 application is to continue to investigate the preclinical use of antiretroviral nanoparticles (AR NPs) as a weekly prevention strategy based on the previous R15 and R56 awards. Tenofovir disoproxil fumurate, TDF will be used along with other drugs (elvitegravir or rilpivirine) and fabricated into polymeric nanoparticles, (NPs). The polymeric NPs will be freeze-dried and reconstituted with 5% dextrose for subcutaneous (SubQ) administration as a long-acting drug delivery system. This application is designed to answer several questions regarding dissemination of combinations of anti-HIV drugs using in vitro and in vivo systems. Mice will be used to determine female reproductive tract (FRT) tissue drug and active metabolite levels using LC-MS/MS analysis. Ultimately, hu-BLT mice will be used to determine efficacy of the optimal doses of combination antiretroviral NPs. The specific aims are as follows. Specific Aim 1 will involve the fabrication o single or combinations of 2 antiretroviral drugs (TDF, elvitegravir, or rilpivirine) and test these polymeric NPs in vitro using cell culture systems. Specific Aim 2 will investigate 3 separate doses and tissue pharmacokinetics (PK) of these single or combination drugs from the polymeric NPs in reproductive tissues from NSG mice for both antiretroviral levels and TDF active metabolite using LC-MS/MS analysis. The first 2 years will be used to determine vaginal tissue concentrations that will be > 90 ng/mg for the drug(s) contained in the NPs at 7 days post SubQ administration as the go-no go determinant. Specific Aim 3 will be to apply the most optimal polymeric NP doses of combination NPs in the hu-BLT mouse model of HIV. It is our intention to determine in the hu-BLT mouse model whether these NPs are able to protect mice from HIV-1 infection over 7-days. The results of this application should offer a new long-acting formulation for SubQ administration that contains combinations of antiretroviral drugs fabricated into polymeric NPs. The weekly SubQ administration of polymeric NPs in hu-BLT mice will translate using allometry into monthly administration in humans.